We previously identified an ensemble of proteasome components which had been largely unrecognized for years because they are released from proteasomes by the high salt used in conventional purifications. We now propose to focus more narrowly on two such factors: Hul5, an ubiquitin-protein ligase, and Ubp6, a deubiquitinating enzyme. We have found that Ubp6 has a second, highly unexpected function: noncatalytic inhibition of protein degradation. Moreover, when the proteasome is inhibited by UbpG, the other deubiquitinating enzyme of the proteasome, Rpn11, is suppressed. Since Ubp6 trims ubiquitin chains progressively, while Rpn11 removes chains en bloc, the presence of Ubp6 imposes a switch in the mode of substrate deubiquitination. Interestingly, Ubp6 was also found to be regulated through a novel ubiquitin stress response. We found that Hul5 serves to extend the chains of proteasome-bound ubiquitin conjugates, acting in particular on degradative substrates such as cyclin B. Accordingly, hu!5 mutants show stabilization of many substrates. Hul5 binding to proteasomes is enhanced by UbpG, and functional opposition between them is suggested by suppression of ubp6 by the hul5 null mutation. Moreover, products of Hul5 conjugation can be rapidly disassembled by proteasome-bound Ubp6. We propose that, by regulating the lengths of proteasome-bound ubiquitin chains, the balance of Hul5 and Ubp6 activity regulates substrate commitment to degradation. The degradation delay caused by Ubp6 might serve to ensure that the deubiquitinating activity of Ubp6 and the ubiquitin-ligating activity of Hul5 have adequate time to refashion substrate-bound ubiquitin chains. We will more fully characterize noncatalytic proteasome inhibition by Ubp6 with the benefit of a quantitative degradation assay, and determine the mechanism of inhibition. We recently found that Hul5 also has a prominent noncatalytic influence on proteasome function, and this will be further characterized. We will also test the following hypotheses: that UbpG's activity of deubiquitination also mediates proteasome inhibition;and that there is a functional opposition between Hul5 and Ubp6, resulting in ubiquitin chains being in a dynamic state when bound to the proteasome. The proteasome is a key cellular regulator and is important for human health, as proteasome inhibitors are in clinical use in the treatment of a growing list of cancers. Thus, by providing a deeper understanding of this enzyme, our studies may facilitate the development of other useful drugs.